U.S. patent number 4,888,285 [Application Number 07/129,163] was granted by the patent office on 1989-12-19 for enzyme immobilization on a water-insoluble amino group-containing carrier.
This patent grant is currently assigned to Hitachi, Ltd.. Invention is credited to Ryoichi Haga, Masahiko Ishida, Yuusaku Nishimura.
United States Patent |
4,888,285 |
Nishimura , et al. |
December 19, 1989 |
Enzyme immobilization on a water-insoluble amino group-containing
carrier
Abstract
An immobilized enzyme having high activity and stability is
obtained by immobilizing an enzyme on a water-insoluble amino
group-containing carrier by use of a polyfunctional cross-linking
agent such as glutaraldehyde in the presence of a phenolic
carboxylic acid having one or more hydroxyl groups such as tannic
acid. In addition to the phenolic carboxylic acid, a basic
polysaccharide such as chitosan may also be present. The amino
group-containing carrier may be aminated silica gel, aminated
porous glass, aminated zeolite, or water insoluble crosslinked
chitosen.
Inventors: |
Nishimura; Yuusaku (Hitachi,
JP), Ishida; Masahiko (Hitachi, JP), Haga;
Ryoichi (Hitachi, JP) |
Assignee: |
Hitachi, Ltd. (Tokyo,
JP)
|
Family
ID: |
17755955 |
Appl.
No.: |
07/129,163 |
Filed: |
December 7, 1987 |
Foreign Application Priority Data
|
|
|
|
|
Dec 8, 1986 [JP] |
|
|
61-290432 |
|
Current U.S.
Class: |
435/176; 435/177;
435/178 |
Current CPC
Class: |
C12N
11/10 (20130101); C12N 11/14 (20130101); C12N
11/06 (20130101) |
Current International
Class: |
C12N
11/10 (20060101); C12N 11/06 (20060101); C12N
11/14 (20060101); C12N 11/00 (20060101); C12N
011/14 (); C12N 011/02 (); C12N 011/10 () |
Field of
Search: |
;435/174,176,177,178,180,181,182 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Naff; David M.
Attorney, Agent or Firm: Fay, Sharpe, Beall, Fagan, Minnich
& McKee
Claims
What we claim is:
1. A method for immobilizing an enzyme on a water-insoluble carrier
comprising the steps of:
(a) adding a phenolic carboxylic acid having one or more hydroxyl
groups to an aqueous solution containing an enzyme;
(b) permitting said enzyme to be absorbed on said phenolic
carboxylic acid;
(c) adding to the solution a water-insoluble amino group-containing
carrier and a polyfunctional crosslinking agent;
(d) permitting said enzyme absorbed on said phenolic carboxylic
acid to bind with said water-insoluble amino group-containing
carrier through said polyfunctional crosslinking agent; and,
(e) recovering the resultant immobilized enzyme on a
water-insoluble carrier from the aqueous solution.
2. The method for immobilization of enzyme according to claim 1,
wherein the water-insoluble carrier containing amino groups is
aminated silica gel, aminated porous glass, aminated zeolite, or
water-insoluble crosslinked chitosan.
3. The method for immobilization of enzyme according to claim 1,
wherein the polyfunctional crosslinking agent is a
polyaldehyde.
4. The method for immobilization of enzyme according to claim 3,
wherein the polyaldehyde is glutaraldehyde.
5. The method for immobilization of enzyme according to claim 1,
wherein the phenolic carboxylic acid is tannic acid, gallic acid or
catechol.
6. The method for immobilization of enzyme according to claim 1,
wherein the phenolic carboxylic acid is tannic acid.
7. A method for immobilizing an enzyme on a water-insoluble carrier
comprising the steps of:
(a) adding a phenolic carboxylic acid having one or more hydroxyl
groups and a basic polysaccharide having high adsorbability for
enzymes to an aqueous solution containing an enzyme;
(b) permitting said enzyme to be absorbed on said phenolic
carboxylic acid and said basic polysaccharide;
(c) adding to the solution a water-insoluble amino group-containing
carrier and a polyfunctional crosslinking agent;
(d) permitting said enzyme absorbed on said phenolic carboxylic
acid and said basic polysaccharide to bind with said
water-insoluble amino group-containing carrier through said
polyfunctional crosslinking agent; and,
(e) recovering the resultant immobilized enzyme on a
water-insoluble carrier from the aqueous solution.
8. The method for immobilization of enzyme according to claim 7,
wherein the basic polysaccharide having high adsorbability for
enzymes is chitosan.
9. The method for immobilization of enzyme according to claim 7,
wherein the water-insoluble carrier containing amino groups is
aminated silica gel, aminated porous glass, aminated zeolite, or
water-insoluble crosslinked chitosan.
10. The method for immobilization of enzyme according to claim 7,
wherein the polyfunctional crosslinking agent is
glutaraldehyde.
11. The method for immobilization of enzyme according to claim 7,
wherein the phenolic carboxylic acid is tannic acid.
12. An immobilized enzyme produced by a method for immobilizing an
enzyme on a water-insoluble carrier comprising the steps of:
(a) adding a phenolic carboxylic acid having one or more hydroxyl
groups to an aqueous solution containing an enzyme;
(b) permitting said enzyme to be absorbed on said phenolic
carboxylic acid;
(c) adding to the solution a water-insoluble amino group-containing
carrier and a polyfunctional crosslinking agent;
(d) permitting said enzyme absorbed on said phenolic carboxylic
acid to bind with said water-insoluble amino group-containing
carrier through said polyfunctional crosslinking agent; and,
(e) recovering the resultant immobilized enzyme on a
water-insoluble carrier from the aqueous solution.
13. The immobilized enzyme according to claim 12, wherein the
water-insoluble carrier is aminated silica gel, aminated porous
glass, aminated zeolite, or water-insoluble crosslinked
chitosan.
14. The immobilized enzyme according to claim 12, wherein the
polyfunctional crosslinking agent is glutaraldehyde.
15. The immobilized enzyme according to claim 12, wherein the
phenolic carboxylic acid is tannic acid.
16. An immobilized enzyme produced by a method for immobilizing an
enzyme on a water-insoluble carrier comprising the steps of:
(a) adding a phenolic carboxylic acid having one or more hydroxyl
groups and a basic polysaccharide having high adsorbability for
enzymes to an aqueous solution containing an enzyme;
(b) permitting said enzyme to be absorbed on said phenolic
carboxylic acid and said basic polysaccharide;
(c) adding to the solution a water-insoluble amino group-containing
carrier and a polyfunctional crosslinking agent;
(d) permitting said enzyme absorbed on said phenolic carboxylic
acid and said basic polysaccharide to bind with said
water-insoluble amino group-containing carrier through said
polyfunctional crosslinking agent; and,
(e) recovering the resultant immobilized enzyme on a
water-insoluble carrier from the aqueous solution.
17. The immobilized enzyme according to claim 16, wherein the basic
polysaccharide having high adsorbability for enzymes is
chitosan.
18. The immobilized enzyme according to claim 16, wherein the
water-insoluble carrier is aminated silica gel, aminated porous
glass, aminated zeolite, or water-insoluble crosslinked
chitosan.
19. The immobilized enzyme according to claim 16, wherein the
polyfunctional crosslinking agent is glutaraldehyde.
20. The immobilized enzyme according to claim 16, wherein the
phenolic carboxylic acid is tannic acid.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a method for immobilizing the enzyme on a
water-insoluble carrier and to an immobilized enzyme produced
thereby.
More particularly, this invention relates to a method for
immobilizing an enzyme on a water-insoluble amino group-containing
carrier such as silica gel, porous glass or the like having amino
groups introduced thereinto, by use of a polyfunctional
crosslinking agent in the presence of a phenolic carboxylic acid
having one or more hydroxyl groups such as tannic acid or the like
or in the presence of a phenolic carboxylic acid having one or more
hydroxyl groups and chitosan. In addition, the present invention is
also directed to the immobilized enzymes obtained by said
method.
According to this invention, an enzyme can be stably immobilized on
a water-insoluble carrier while retaining its high activity,
thereby producing an immobilized enzyme which permits continuous
enzymatic reactions for a long period of time. Through the use of
the immobilized enzyme of this invention, the purity of a product
obtained by enzymatic reaction can be improved, the amount of
enzyme used can be reduced, and the enzymatic reaction vessel
required for the enzymatic process can be made more compacted.
2. Related Art Statement
Enzymatic reactions are widely used in industrial production
processes of medicines, foods etc. Such enzymatic reactions have
heretofore been carried out in solutions prepared by dissolving an
enzyme in an aqueous solution of substrate. However, such methods
require not only very complicated procedures, for example, the
steps of supplying fresh enzyme while keeping the reaction
conditions constant and of recovering the enzyme after the
reaction, but also very troublesome procedures of separating and
purifying the reaction product. In order to remove these defects,
continuous enzymatic reactions directed to the use of an enzyme
immobilized on a water-insoluble carrier have been investigated in
recent years.
Along this line, the main methods for immobilizing enzymes include:
known, for example, (1) the carrier covalent binding method, (2)
the physical adsorption method or the ionic binding method, (3) the
crosslinking method and (4) the entrapping method.
Among these immobilization methods, the carrier covalent binding
method of (1) permits production of an immobilized enzyme in which
the binding power between the carrier and the enzyme is relatively
strong, but is disadvantageous in that the determination of optimal
conditions for the covalent binding reaction is difficult, and that
the production of an immobilized enzyme having high activity is
also generally difficult. The physical adsorption method or ionic
binding method of (2) permits immobilization of an enzyme by a
simple procedure under mild conditions and hence the production of
an immobilized enzyme having relatively high activity, but is
disadvantageous in that since the binding power between the carrier
and the enzyme is weak, the enzyme tends to be released from the
carrier. The crosslinking method of (3) is disadvantageous in that
since the crosslinking reaction is carried out under relatively
severe conditions, only an immobilized enzyme having low activity
can be obtained. The entrapping method of (4) is generally
advantageous in that an immobilized enzyme can be produced easily
at low cost, but is disadvantageous not only in that when a
water-soluble polymer such as polyacrylamide is crosslinked and
then gelatinized for entrapping an enzyme, the enzymic activity is
unavoidably lowered, but also in that the enzyme is liable to be
released from the gel formed.
As the method for immobilizing the enzyme according to the carrier
covalent binding method of (1), there may be exemplified, for
example, a method which comprises forming Schiff bases or peptide
bonds between the functional groups (--NH.sub.2, --COOH, etc.) on
the surface of a carrier and the --NH.sub.2, --COOH, etc. groups
present in the enzyme molecules by use of a polyfunctional
crosslinking agent or a condensing agent. A method comprising
activating an amino group-containing carrier by use of a
crosslinking agent such as glutaraldehyde, and then immobilizing
glucoamylase thereon is described in Starch/Stark 33 (1981) Nr. 2,
S.52-55. Further, immobilization of glucoamylase on aminated silica
gel by use of glutaraldehyde is described in Enzyme Microb.
Technol., 1982, Vol. 4, Mar. pp. 89-92. However, the half life of
the enzymic activity obtained by these methods is short, so that
the period of time of stable saccharification produced by these
immobilized enzymes is also short. Therefore, continuous
saccharification using these immobilized enzymes cannot be carried
out for a long period of time unless there is employed, for
example, a method comprising placing a large number of reaction
tanks in parallel and using them one after another. In this case,
complicated apparatuses and procedures are required and the enzyme
should be frequently renewed.
As another carrier covalent binding method, there is a method which
comprises immersing a molded product of swollen chitin in an enzyme
solution, then carrying out glutaraldehyde treatment, thereby
producing an immobilized enzyme (Japanese Patent Application Kokai
(Laid-Open) No. 111686/86). However in the case of such a method,
it is also difficult to obtain a stably immobilized enzyme
retaining high activity.
As described above, the immobilization processes of the prior art,
in particular, the carrier covalent binding method, have many
disadvantageous, for example, insufficient consideration has been
given to the affinity of the carrier surface for the enzyme,
determination of conditions for covalent binding reaction is
complicated, and it is difficult to obtain an immobilized enzyme
having high activity.
On the other hand, methods for producing an immobilized enzyme by
the crosslinking method include, for example, a method comprising
the steps of coagulating a cell enzyme by use of a macromolecule
coagulant such as chitosan, and then carrying out a crosslinking
reaction through the use of glutaraldehyde or the like to
immobilize the cell enzyme (Japanese Patent Application Kokai
(Laid-Open) No. 120182/77); a method comprising using tannin
together with a coagulant such as polyethyleneimine, and then
immobilizing an enzyme by use of a crosslinking agent such as
glutaraldehyde (Japanese Patent Application Kokai (Laid-Open) No.
110190/82); and a method comprising the steps of using
polyethyleneimine as a coagulant, carrying out a crosslinking
reaction in the presence of chitosan through the use of
glutaraldehyde, thereby immobilizing an enzyme (Japanese Patent
Application Kokai (Laid-Open) No. 58072/85). These methods use
chitosan or tannin, but in all of them, immobilized enzymes are
obtained by the crosslinking methods. Thus, they are different from
the carrier covalent binding method in which an enzyme is
immobilized on a water-soluble carrier.
SUMMARY OF THE INVENTION
An object of this invention is to provide an immobilized enzyme
having high activity and stability by simple covalent binding
reaction of the surface of a water-insoluble carrier with an
enzyme.
Another object of this invention is to provide a method for the
immobilization of an enzyme wherein the enzyme is stably
immobilized on a water-insoluble amino group-containing carrier
while retaining its high activity.
Other and further objects and advantages of this invention will be
apparent from the following description.
The above objects and advantages of this invention can be achieved
by carrying out the covalent binding reaction of an enzyme with the
surface of a carrier containing amino groups as surface functional
groups in the presence of a substance having specific adsorbability
for enzymes, i.e., a phenolic carboxylic acid having one or more
hydroxyl groups, preferably in the presence of a phenolic
carboxylic acid having one or more hydroxyl groups and a basic
polysaccharide having high adsorbability for enzymes such as
chitosan, and thereby obtaining an immobilized enzyme.
That is to say, a first feature of the invention is a method for
the immobilization of an enzyme which comprises immobilizing an
enzyme in an aqueous solution containing the enzyme on a
water-insoluble carrier having amino groups in the molecule, said
immobilization being carried out by covalent binding reaction of
the enzyme with the water-insoluble carrier through a
polyfunctional crosslinking agent in the presence of a phenolic
carboxylic acid having one or more hydroxyl groups.
A second feature of the invention is a method for the
immobilization of an enzyme which comprises immobilizing an enzyme
in an aqueous solution containing the enzyme on a water-insoluble
carrier containing amino groups in the molecule, said
immobilization being carried out by covalent binding reaction of
the enzyme with the carrier through a polyfunctional crosslinking
agent in the presence of a phenolic carboxylic acid having one or
more hydroxyl groups and a basic polysaccharide having high
adsorbability for enzymes.
A third feature of the invention is a method for the immobilization
of an enzyme which comprises forming Schiff bases between a
water-insoluble carrier having amino groups in the molecule and an
enzyme in an aqueous solution containing the enzyme by use of a
polyfunctional crosslinking agent, and thereby immobilizing the
enzyme, said Schiff base formation being carried out in the
presence of a phenolic carboxylic acid having one or more hydroxyl
groups.
A fourth feature of the invention is a method for the
immobilization of an enzyme which comprises forming Schiff bases
between a water-insoluble carrier having amino groups in the
molecule and an enzyme in an aqueous solution containing the enzyme
by use of a polyfunctional crosslinking agent, and thereby
immobilizing the enzyme, said Schiff base formation being carried
out in the presence of a phenolic carboxylic acid having one or
more hydroxyl groups and a basic polysaccharide having high
adsorbability for enzymes.
A fifth feature of the invention is an immobilized enzyme obtained
by the immobilization on a water-insoluble carrier by use of a
polyfunctional crosslinking agent, said immobilization being
carried out by adsorption of an enzyme on a phenolic carboxylic
acid having one or more hydroxyl groups, followed by covalent
binding reaction of the amino groups of the water-insoluble carrier
and the amino groups of the enzyme with the functional groups of
the crosslinking agent.
A sixth feature of the invention is an immobilized enzyme obtained
by the immobilization on a water-insoluble carrier by use of a
polyfunctional crosslinking agent, said immobilization being
carried out by adsorption of an enzyme on a phenolic carboxylic
acid having one or more hydroxyl groups and a basic polysaccharide
having high adsorbability for enzymes, followed by covalent binding
reaction of the amino groups of the water-insoluble carrier and the
amino groups of the enzyme with the functional groups of the
crosslinking agent.
A seventh feature of the invention is an immobilized enzyme
obtained by the immobilization on a water-insoluble carrier by use
of a polyfunctional crosslinking agent, said immobilization being
carried out by adsorption of an enzyme on a phenolic carboxylic
acid having one or more hydroxyl groups, followed by formation of
Schiff bases between the functional groups of the crosslinking
agent and the amino groups of the water-insoluble carrier and of
the enzyme.
An eighth feature of the invention is an immobilized enzyme
obtained by the immobilization on a water-insoluble carrier by use
of a polyfunctional crosslinking agent, said immobilization being
carried out by adsorption of an enzyme on a phenolic carboxylic
acid having one or more hydroxyl groups and a basic polysaccharide
having high adsorbability for enzymes, followed by formation of
Schiff bases between the functional groups of the crosslinking
agent and the amino groups of the water-insoluble carrier and of
the enzyme.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing the relationship between glutaraldehyde
concentration and the activity of a carrier having glucoamylase
immobilized thereon.
FIG. 2 is a bar graph showing comparison of the activities of
carriers having glucoamylase immobilized thereon which were
obtained by immobilization on aminated silica gel by various
methods.
FIG. 3 is a graph showing the thermostability of carriers having
glucoamylase immobilized thereon which were obtained by various
methods.
FIG. 4 is a graph showing the test results of continuous
saccharification of dextrin by carriers having glucoamylase
immobilized thereon which were obtained by various methods.
DETAILED DESCRIPTION OF THE INVENTION
The water-insoluble amino group-containing carrier used in this
invention includes, for example, water-insoluble aminated carriers
obtained by introducing amino groups into silica gels, porous
glasses, zeolites, etc. through silane coupling reaction using an
aminosilane derivative, and amino polysaccharide carriers obtained
by crosslinking chitosan formed by deacetylation of chitin and
thereby insolubilizing the same.
As a method for covalent binding of an enzyme to amino groups which
are functional groups on the surface of said water-insoluble
carrier, a method comprising forming Schiff bases between these
amino groups and the amino groups in the enzyme is preferred. When
glutaraldehyde is used as a polyfunctional crosslinking agent, the
formation of the Schiff bases is represented by the formula (1)
shown below. The polyfunctional agent includes polyaldehydes such
as glutaraldehyde, dialdehyde starch, malonaldehyde, succinic
aldehyde, etc. Among them, glutaraldehyde is particularly
preferred. ##STR1##
In this invention, an enzyme is immobilized in the presence of a
substance having specific adsorbability for enzymes. Such a
substance includes, for example, phenolic carboxylic acids having
one or more hydroxyl groups such as tannic acid, pyrogallol tannin,
gallic acid, catechol and the like. Among the phenolic carboxylic
acids, tannic acid is particularly preferred. In this invention, it
is preferable to use basic polysaccharides having high
adsorbability for enzymes, together with the phenolic carboxylic
acids. As such polysaccharides, chitosan which is a chitin
derivative is exemplified.
As the enzyme used in this invention, any enzyme may be used so
long as it is adsorbed on tannic acid, chitosan or the like without
losing its activity. Said enzyme includes, for example,
glucoamylase, .beta.-amylase, .alpha.-amylase, glutamate
decarboxylase, glucose isomerase and inulase.
A desired immobilized enzyme is preferably obtained by placing an
enzyme-containing aqueous solution together with a water-insoluble
amino group-containing carrier, a polyfunctional crosslinking
agent, and a phenolic carboxylic acid having one or more hydroxyl
groups alone or a combination of a phenolic carboxylic acid having
one or more hydroxyl groups and chitosan, and carrying out the
reaction at 5.degree. to 40.degree. C. for several hours.
The shape of the water-insoluble amino-group-containing carrier is
preferably granular or bead-like, and its size is preferably about
0.1 to about 5 mm, particularly preferably about 0.2 to about 0.5
mm. When the size is too large, the void volume is increased, so
that the enzymic activity per volume is decreased. On the other
hand, when the size is too small, the pressure loss becomes too
large or separation of the immobilized enzyme from the reaction
mixture becomes difficult. Therefore, it is not desirable.
The pH of the reaction mixture at the time of enzyme immobilization
is in the range where the enzyme is not deactivated. The reaction
temperature is in the range where no thermal deactivation occurs,
and is, in practice, preferably in the range of 5.degree. to
40.degree. C. The reaction time is 1 to 10 hours. When the reaction
temperature is high, the reaction time may be short and shaking or
stirring is recommendable for carrying out the reaction
efficiently. At the time of immobilization, the amount of the
solution containing an enzyme and the like is 5 to 10 times that of
the carrier. When the amount of the solution is too small, the
carrier tends to be broken by shaking or stirring at the time of
the reaction. On the other hand, the amount of the solution is too
large, the rate of immobilization reaction is decreased.
In the invention, the amount of the polyfunctional crosslinking
agent used for the enzyme immobilization is preferably
stoichiometrical with regard to the amino groups of the carrier. In
the invention, as described hereinafter, the polyfunctional
crosslinking agent need not be used in excess, but even when it is
used in excess, an immobilized enzyme having sufficiently high
activity can be obtained.
The amount of the phenolic carboxylic acid used for the enzyme
immobilization is usually about 0.1% by weight or more, preferably
about 0.1% to about 10% by weight based on the weight of the
enzyme-containing aqueous solution.
The amount of the basic polysaccharide having high adsorbability
for enzymes such as chitosan or the like is usually preferably
about 0.01% to about 0.1% by weight based on the weight of the
enzyme-containing aqueous solution.
In order to obtain an immobilized enzyme having high activity, it
is preferable to remove enzyme which is liable to be released
because of insufficient binding, the surplus polyfunctional
crosslinking agent, and the enzyme adsorbents such as tannic acid,
chitosan, etc. by washing a carrier having enzyme immobilized
thereon, sufficiently with a suitable buffer solution after
reaction of the polyfunctional crosslinking agent.
In a method comprising forming Schiff bases between the amino
groups on the surface of a water-insoluble amino group-containing
carrier and the amino groups in enzyme molecules by use of a
polyfunctional crosslinking agent such as glutaraldehyde or the
like, and thereby immobilizing the enzyme, glutaraldehyde or the
like as polyfunctional crosslinking agent should be present
generally in a stoichiometric or larger amount for rapid and
complete progress of the Schiff base formation reaction. However,
it is difficult to obtain an immobilized enzyme having high
activity by such an immobilization method because when a large
amount of a polyfunctional crosslinking agent is present, a part of
enzyme molecules to be immobilized are chemically modified, so that
the higher-order structure of the active site of the protein is
broken resulting in deactivation of the enzyme. Therefore, in
conventional immobilization methods, there is unavoidably employed
a method which comprises previously treating an amino
group-containing carrier with an excess of polyfunctional
crosslinking agent to activate the carrier, removing the unreacted
polyfunctional crosslinking agent, and then reacting an enzyme with
the carrier to immobilize the same thereon. But this method is
disadvantageous in that since crosslinking reaction of the amino
groups of the carrier with one another proceeds considerably and
hence functional groups which react with an enzyme to be
immobilized are decreased in number, the amount of enzyme
immobilized is decreased, so that the activity of the resulting
carrier having enzyme immobilized thereon is generally low.
On the other hand, it has been found that when as in this
invention, covalent binding reaction of the amino groups of a
carrier with an enzyme by use of a polyfunctional crosslinking
agent is carried out in the presence of a substance having specific
adsorbability for enzymes, for example, a phenolic carboxylic acid
having one or more hydroxyl groups such as tannic acid or the like
or chitosan together therewith, deactivation of the enzyme can be
prevented, so that an immobilized enzyme having high activity and
stability can be obtained, even when the polyfunctional
crosslinking agent is present in excess.
The reason why the immobilized enzyme of this invention obtained by
using a phenolic carboxylic acid having one or more hydroxyl groups
has high activity seems to be as follows. The phenolic carboxylic
acid which has specific adsorbability for enzymes adsorbs a larger
amount of enzyme to stabilize the higher-order structure of this
enzyme, so that deactivation by the polyfunctional crosslinking
agent is prevented. Moreover, the amino groups of the carrier and
the amino groups of the enzyme are linked to each other through the
polyfunctionals groups of the polyfunctional crosslinking agent, so
that the enzyme is strongly immobilized on the carrier.
The reason why the immobilized enzyme of this invention obtained by
using a basic polysaccharide together with a phenolic carboxylic
acid having one or more hydroxyl groups, preferably chitosan, has
still higher activity seems to be as follows. Chitosan is a basic
polysaccharide and has high adsorbability for enzymes, and
therefore when chitosan is present together with a phenolic
carboxylic acid having one or more hydroxyl groups, enzyme is
adsorbed on the both in common, so that its higher-order structure
becomes still stronger.
As described above in detail, according to this invention, enzyme
can be immobilized on a water-insoluble carrier stably while
retaining its high activity, and there can be obtained an
immobilized enzyme which permits continuous enzymatic reaction for
a long period of time. Therefore, by use of the immobilized enzyme
of this invention, the purity of a product obtained by enzymatic
reaction can be improved, the amount of enzyme to be used can be
reduced, and an enzymatic reaction vessel can be made more
compacted.
This invention is concretely illustrated below with reference to an
Example for the a method for immobilization of glucoamylase derived
from Aspergillus niger which is used mainly for the
saccharification of starch. In addition, this invention is
concretely illustrated with reference to Examples for methods for
the immobilization of .alpha.-amylase and glutamate decarboxylase,
respectively.
EXAMPLE 1
Immobilization of glucoamylase
Silica gel (particle diameter 0.3 .phi., pore diameter 500.ANG.)
was aminated with .gamma.-aminotriethoxysilane in toluene to
prepare a carrier (hereinafter referred to SiO.sub.2 --NH.sub.2 in
some cases), and 1 ml of the carrier was kept in contact with 10 ml
of an aqueous solution (0.05 M acetate buffer, pH 4.5) containing
0.5 ml of glucoamylase (3000 U/ml), at room temperature for 4 hours
to carry out immobilization. Then, the thus treated carrier was
washed with 0.05 M acetate buffer (pH 4.5) to remove the surplus
glucoamylase, whereby immobilized glucoamylase was obtained. Stable
immobilization was variously attempted by properly adding
glutaraldehyde, tannic acid (in an amount of 1.0% by weight based
on the weight of the glucoamylase-containing aqueous solution),
chitosan (in an amount of 0.05% by weight based on the weight of
the glucoamylase-containing aqueous solution), etc. alone or in
combination of two or more thereof at the time of the
immobilization reaction.
Determination of the Activity of Carriers Having Glucoamylase
Immobilized Thereon
15 ml of a 30% aqueous dextrin solution (pH 4.5) was kept in
contact, under shaking at 60.degree. C., with 1 ml of each of
carriers having glucoamylase immobilized thereon which had been
obtained by carrying out immobilization reaction in the presence or
absence of tannic acid by use of various concentrations of
glutaraldehyde or with 1 ml of each of carriers having glucoamylase
immobilized thereon which had been obtained in the presence of
tannic acid or gallic acid by or without using glutaraldehyde.
Then, the concentration of glucose produced was measured, whereby
the relative activity of each carrier having glucoamylase
immobilized thereon was determined. The proportion of reducing
sugar of the dextrin used as substrate was 18%.
The results obtained are shown in FIG. 1 and FIG. 2.
FIG. 1 shows the relationship between glutaraldehyde concentration
and the activity of an immobilized product in the case where
glucoamylade was immobilized on aminated silica gel by using a
polyfunctional crosslinking agent glutaraldehyde. When tannic acid
which is one of the phenolic carboxylic acids is present in the
immobilization reaction, an immobilized product having high
activity can be obtained in a wide glutaraldehyde concentration
range. On the other hand, when tannic acid is absent, the activity
of immobilized product is seriously affected by the glutaraldehyde
concentration, and the optimum glutaraldehyde concentration range
is very narrow. In addition, the activity of immobilized product is
sharply lowered with an increase of the glutaraldehyde
concentration. As it generally known, most enzymes are denatured by
organic solvents to be deactivated. The reason why the activity of
the immobilized product obtained in the absence of tannic acid is
low is that glucoamylase is deactivated by glutaraldehyde. From the
above, it can be seen that in this invention, the optimum
glutaraldehyde concentration is stoichiometrical with regard to the
amino groups of a carrier, as shown by the formula (1) mentioned
hereinbefore. For preventing the deactivation of glucoamylase by
glutaraldehyde, it was sufficient that tannic acid was present in a
very small amount of, preferably about 0.1 per cent by weight, more
preferable several per cent by weight based on the weight of the
glucoamylase-containing aqueous solution. It was found that when
the covalent binding reaction of an aminated carrier with
glucoamylase by use of glutalaldehyde was carried out in the
presence of tannic acid, the enzyme was not deactivated and even
when a sufficient amount of glutaraldehyde was used, the reaction
proceeded rapidly and completely, so that there could be obtained a
carrier having glucoamylase immobilyzed thereon which had high
activity and stability.
FIG. 2 shows comparison of the activities of carriers having
glucoamylase immobilized thereon which were obtained by
immobilizing glucoamylase on aminated silica gel (SiO.sub.2
--NH.sub.2) by various immobilization methods. It can be seen that
in the case of a method using a phenolic carboxylic acid having one
or more hydroxyl groups such as tannic acid, gallic acid, etc., the
activity is higher than in the case of a conventional method using
glutaraldehyde (GA) alone.
Theremostability Test on Carriers Having Glucoamylase Immobilized
Thereon
1 ml of each carrier having glucoamylase immobilized thereon which
had been obtained using tannic acid or catechol was kept in contact
with 15 ml of a 30% aqueous glucose solution (pH 4.5) at 60.degree.
C., and after heat treatment, the residual activity of this
immobilized product was measured.
The results obtained were as shown in FIG. 3.
FIG. 3 shows the thermostability of carriers having glucoamylase
immobilized thereon. The activity of the immobilized products
obtained by use of a phenolic carboxylic acid having one or more
hydroxyl groups such as tannic acid or catechol is high. But
employment of a phenolic carboxylic acid having one or more
hydroxyl groups alone is not desirable because glucoamylase
adsorbed thereon is merely deposited on the surface of a carrier,
so that glucoamylase is easily released by longtime heat-treatment,
resulting in a rapid lowering of the residual activity.
On the other hand, in the case of a conventional immobilized
product obtained by use of glutaraldehyde alone, glucoamylase is
immobilized by covalent binding to the amino groups of a carrier,
so that glucoamylase is not easily released, and its deactivation
mainly accompanies thermal denaturation and the lowering of the
residual activity by heat treatment is appreciably lessened. On the
other hand, in the case of the immobilized product of this
invention obtained by use of a phenolic carboxylic acid having one
or more hydroxyl groups and glutaraldehyde, the lowering of the
residual activity is still slighter, indicating that this
immobilized product is excellent in thermostability.
Continuous Saccharification of Dextrin
A column (inside diameter: 16 .phi.) maintained at 55.degree. C.
was packed with 20 ml of each carrier having glucoamylase
immobilized thereon which had been obtained using tannic acid alone
or a combination of tannic acid and chitosan, and a 30% aqueous
dextrin solution (pH 4.5) was passed therethrough at a flow rate of
1 ml/min, whereby continuous saccharification of dextrin was
carried out.
The results obtained are shown in FIG. 4.
FIG. 4 shows test results of continuous saccharification of dextrin
by each carrier having glucoamylase immobilized thereon. The stable
saccharification time in the case of immobilized glycoamylase
obtained by immobilization using glutaraldehyde together with
tannic acid which is one of the phenolic carboxylic acids is about
twice that in the case of immobilized glucoamylase obtained by
glutaraldehyde alone according to a conventional method.
Furthermore, the stable saccharification time in the case of
immobilized glucoamylase obtained by using chitosan, a chitin
derivative in combination with glutaraldehyde and tannic acid is
about 3 times that in the case of the latter immobilized
glucoamylase.
In addition, when tannic acid alone is used together with
glutaraldehyde, the environment of enzyme is an acidic atmosphere,
while when a combination of chitosan and tannic acid is used
together with glutaraldehyde, the environment of enzyme becomes a
neutral atmosphere and this condition seems to have a beneficial
effect on the thermostability of immobilized glucoamylase. Although
the adding amount of chitosan depends on the immobilization
conditions, a sufficient effect can be obtained when the adding
amount is about 0.1% by weight based on the weight of the
glucoamylase-containing aqueous solution.
EXAMPLE 2
To 1 ml of aminated silica gel was added 10 ml of an aqueous
solution (pH 7.0) containing 0.5 ml of .alpha.-amylase, followed by
adding thereto tannic acid in an amount of 1% by weight based on
the weight of the .alpha.-amylase-containing aqueous solution and
glutaraldehyde in an amount of 0.5% by weight based on the weight
of the .alpha.-amylase-containing aqueous solution. The aminated
silica gel was thus kept in contact with the
.alpha.-amylase-containing aqueous solution at room temperature for
4 hours to immobilize .alpha.-amylase.
The activity of the immobilized .alpha.-amylase was determined in
the following manner, and the initial activity and the half life of
activity were evaluated.
With 15 ml of a 30% aqueous potato starch solution (pH 7, Ca.sup.++
3 mM) was kept in contact 1 ml of the immobilized .alpha.-amylase
at 80.degree. C. for 20 minutes, and the reducing sugars produced
were determined.
The half life of activity was defined as time required for the
initial activity to be reduced by 50% in the case where treatment
in an aqueous solution (pH 7.0) at 80.degree. C. was carried
out.
COMPARATIVE EXAMPLE 2
To 1 ml of aminated silica gel was added 10 ml of an aqueous
solution (pH 7.0) containing 0.5 ml of .alpha.-amylase, followed by
adding thereto glutaraldehyde (0.5% by weight). The aminated silica
gel was thus kept in contact with the aqueous solution at room
temperature for 4 hours to immobilized .alpha.-amylase.
EXAMPLE 3
To 1 ml of aminated silica gel was added an aqueous solution (pH
5.0) containing 20 mg of glutamate decarboxylase, followed by
adding thereto tannic acid (1% by weight) and glutaraldehyde (0.5%
by weight). The aminated silica gel was thus kept in contact with
the aqueous solution at room temperature for 2 hours to immobilize
glutamate decarboxylase.
The activity of the immobilized enzyme was determined in the
following manner, and the initial activity and the half life of
activity were evaluated.
With 15 ml of a 0.05 M aqueous glutamic acid solution (pH 5.0) was
kept in contact 1 ml of the immobilized enzyme at 37.degree. C. for
30 minutes, and the .gamma.-aminobutyric acid produced was
determined.
The half life of activity was defined as time required for the
initial activity to be reduced by 50% in the case where treatment
in an aqueous solution (pH 5.0) at 45.degree. C. was carried
out.
COMPARATIVE EXAMPLE 3
To 1 ml of aminated silica gel was added an aqueous solution (pH
5.0) containing 20 mg of glutamate decarboxylase, followed by
adding thereto glutaraldehyde (0.5% by weight). The aminated silica
gel was thus kept in contact with aqueous solution at room
temperature for 2 hours to immobilize glutamate decarboxylase.
EXAMPLE 4
To 1 ml of aminated silica gel was added 10 ml of an aqueous
solution (pH 7.5) containing 50 mg of glucose isomerase, followed
by adding thereto tannic acid (1% by weight) and glutaraldehyde
(0.5% by weight). The aminated silica gel was thus kept in contact
with the aqueous solution at room temperature for 4 hours to
immobilize glucose isomerase.
The activity of the immobilized glucose isomerase was determined in
the following manner, and the initial activity and the half life of
activity were evaluated.
With 15 ml of a 30% aqueous glucose solution (pH 7.5, Mg.sup.++
0.01 M) was kept in contact 1 ml of the immobilized glucose
isomerase at 65.degree. C. for 1 hour, and the fructose produced
was determined.
The half life of activity was defined as time required for the
initial activity to be reduced by 50% in the case where treatment
in an aqueous solution (pH 7.5) at 70.degree. C. was carried
out.
COMPARATIVE EXAMPLE 4
To 1 ml of aminated silica gel was added 10 ml of an aqueous
solution (pH 7.5) containing 50 mg of glucose isomerase, followed
by adding thereto glutaraldehyde (0.5% by weight). The aminated
silica gel was thus kept in contact with the aqueous solution at
room temperature for 4 hours to immobilize glucose isomerase.
EXAMPLE 5
To 1 ml of aminated silica gel was added 10 ml of an aqueous
solution (pH 6.0) containing 100 mg of inulase, followed by adding
thereto tannic acid (1% by weight) and glutaraldehyde (0.5% by
weight). The aminated silica gel was thus kept in contact with the
aqueous solution at room temperature for 4 hours to immobilize
inulase.
The activity of the immobilized inulase was determined in the
following manner, and the initial activity and the half life of
activity were evaluated.
With 15 ml of a 10% aqueous inulin solution (pH 6.0) was kept in
contact 1 ml of the immobilized inulase at 40.degree. C. for 30
minutes, and the fructose produced was determined.
The half life of activity was defined as time required for the
initial activity to be reduced by 50% in the case where treatment
in an aqueous solution (pH 6.0) at 45.degree. C. was carried
out.
COMPARATIVE EXAMPLE 5
To 1 ml of aminated silica gel was added 10 ml of an aqueous
solution (pH 6.0) containing 100 mg of inulase, followed by adding
thereto glutaraldehyde (0.5% by weight). The aminated silica gel
was thus kept in contact with the aqueous solution at room
temperature for 4 hours to immobilize inulase.
The initial activities and the half lives of activity in Examples
2, 3, 4 and 5 and Comparative Examples 2, 3, 4 and 5 are shown in
Table 1. It can be seen from Table 1 that when an enzyme is
immobilized on an amino group-containing carrier by covalent
binding by using a polyfunctional crosslinking agent
glutaraldehyde, the presence of tannic acid is effective in
improving the initial activity and thermostability of the
immobilized enzyme.
TABLE 1 ______________________________________ Initial activity
Half life of activity (relative value) (relative value)
______________________________________ Example 2 100 100
Comparative 81 91 Example 2 Example 3 100 100 Comparative 94 97
Example 3 Example 4 100 100 Comparative 93 69 Example 4 Example 5
100 100 Comparative 76 73 Example 5
______________________________________
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